Method for generating a gene library

ABSTRACT

The present invention relates to a method of generating a gene library from an environmental pool of organisms, which gene library is enriched in DNA encoding a polypeptide with an activity of interest. Also, the invention provides a method of selecting a DNA sequence of interest from an environmental pool of organisms. Further, the invention relates to a gene library prepared from an enriched environmental pool of organisms enriched in DNA encoding a polypeptide with an activity of interest.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation of U.S. Ser. No.09/426,340 (now allowed), which claims priority from U.S. provisionalapplication No. 60/106,319, filed on Oct. 30, 1998, and Danishapplication no. PA 1998 01388, filed on Oct. 28, 1998, which are herebyincorporated by reference.

TECHNICAL FIELD

[0002] The present invention relates to a method of generating a genelibrary from an environmental pool of organisms, which gene library isenriched in DNA encoding a polypeptide with an activity of interest.

BACKGROUND OF THE INVENTION

[0003] The advent of recombinant DNA techniques has made it possible toselect single protein components with interesting properties and producethem on a large scale. This represents an improvement over thepreviously employed production process using microorganisms isolatedfrom nature and producing a mixture of proteins which would either beused as such or separated after the production step. Methods have beendeveloped for rapid identification of genes encoding a polypeptide ofinterest.

[0004] One example is the so called expression cloning techniquedescribed in WO 93/11249 (Novo Nordisk A/S). The technique disclosed inWO 93/11249 comprises a method of screening for a DNA sequence in a DNAlibrary prepared from an organism suspected of producing genes encodingpolypeptides with activities of interest. Such a library hastraditionally been made on DNA isolated from a single knownmicroorganism.

[0005] A compartmentalization method of screening microorganisms havinga selectable characteristic has previously been devised in WO 97/37036,and a process for forming a normalized genomic DNA library from anenvironmental sample is described in Wo 97/37036.

[0006] However, a method of generating a gene library from anenvironmental pool of organisms, which gene library is enriched in DNAencoding a polypeptide with an activity of interest has never beendescribed. Therefore, it would be desirable to have a method based onbiological enrichment for selecting potentially interesting genes fromenvironmental pool of organisms.

SUMMARY OF THE INVENTION

[0007] It has now been found possible to use biological enrichment forselecting potentially interesting genes from an environmental pool oforganisms. Accordingly, the invention provides a method for generating agene library from an environmental pool of organisms, which gene libraryis enriched in DNA encoding a polypeptide with an activity of interest,which method comprises:

[0008] a) subjecting the environmental pool of organisms to cultivationin a medium and/or under conditions suitable for enriching said pool oforganisms in organisms harbouring said DNA; and

[0009] b) preparing a gene library from the resulting enriched pool oforganisms.

[0010] The invention also provides a method of selecting a DNA sequenceof interest from an environmental pool of organisms, which methodcomprises:

[0011] a) subjecting the environmental pool of organisms to cultivationin a medium and/or conditions suitable for enriching said pool oforganisms in organisms harbouring said DNA sequence;

[0012] b) producing gene libraries from the resulting enriched pool oforganisms;

[0013] c) screening the libraries for DNA containing the desired gene;and

[0014] d) selecting the DNA sequence of interest resulting from thescreening of step c).

[0015] Further, the invention relates to a gene library prepared from anenriched environmental pool of organisms enriched in DNA encoding apolypeptide with an activity of interest.

DETAILED DISCLOSURE OF THE INVENTION

[0016] It is an object of the present invention to provide a method forgenerating a gene library from an environmental pool of organisms, whichgene library is enriched in DNA encoding a polypeptide with an activityof interest, which method comprises:

[0017] a) subjecting the environmental pool of organisms to cultivationin a medium and/or under conditions suitable for enriching said pool oforganisms in organisms harbouring said DNA; and

[0018] b) preparing a gene library from the resulting enriched pool oforganisms.

[0019] In the context of the present invention, the term “anenvironmental pool of organisms” means a environmental sample comprisingmicroorganisms and cells from higher animals harboring DNA encoding apolypeptide with an activity of interest. The environmental sample mayfor instance be an environmental sample of soil or plant material,animal or insect dung, insect gut, animal stomach, a marine sample ofsea or lake water, sewage, waste water, a sample of sludge or sediment,etc., comprising one or, as in most case, a vast number of differentmicroorganisms or living cells.

[0020] In step a), the sample as such is cultivated without any need forfurther purification. By selecting the medium and the cultivationconditions at which the sample is cultivated, it is possible forenriching or (amplifying) organisms having optimal growth at thespecific cultivation conditions, and expressing polypeptides withproperties adapted to the cultivation conditions. The gene libraryprepared in step b) may be prepared by any suitable technique known inthe art, non-limiting examples of which are described in Example 3 and4.

[0021] The advantage presented by the present screening method isprimarily that the rate at which novel genes may be isolated and,consequently, novel products be developed may be greatly increased.Furthermore, the method permits screening for multiple polypeptidesactivities and may even result in the isolation of several differentgenes coding for the same type of polypeptides.

[0022] By use of the invention it is possible to exploit enrichedcultures for detecting novel enzymes, and other polypeptides with anactivity of interest.

[0023] In a preferred embodiment, the method of the invention comprisessubjection the environmental pool of organisms to cultivation in amedium, which contains a substrate for the polypeptide with the desiredactivity. A wide range of substrates for the enrichment of theenvironmental of organisms containing different types of gene productsmay be used. For instance, a DNA encoding a polypeptide with an activityof interest such as a pectinase enzyme may be selected as a gene producton a substrate as pectin.

[0024] In a preferred embodiment, the substrate constitutes the carbonsource and/or nitrogen source of the medium.

[0025] In a more preferred embodiment, the substrate comprises pectin,amylose, cellulose, galactan, xylan, arabinan, mannan, lipid orhemicellulose or a combination thereof.

[0026] In a preferred embodiment of the method of the invention, theenrichment is achieved by one or more growth conditions. In a anotherpreferred embodiment, the growth conditions comprise pH and temperature.In yet another preferred embodiment, the growth conditions of step a)used for achieving the enrichment comprises any pH range i.e. 0-12,preferably of about 6-9, in particular 9-12, at any temperature rangei.e. 0-120° C., preferably about 25-30°, preferably 30-50°, mostpreferred 50-70° C.

[0027] An important step in the procedure for selection of a potentiallyinteresting environmental pool of organisms is to select the optimalpool to start from. To select genes encoding polypeptides that can breakdown natural compounds of plant (or animal) origin, it is preferable tolook into those biotopes in nature where such materials are efficientlydecomposed. Examples of animals especially efficient in breaking downplant material are the ruminates, termites and insects (sensu lato)

[0028] In a preferred embodiment, the environmental pool ofmicroorganisms is isolated from an animal stomach or an insect gut.

[0029] In a more preferred embodiment, the pool of microorganisms isisolated from a cow's rumen.

[0030] Likewise, it is important when selecting genes encodingpolypeptides with an activity of interest that are capable of workingunder e.g. strongly alkaline conditions, to isolate the pool oforganisms from an equally strongly alkaline biotope. It is known in theart that in order for Bacillus thuringensis (Bt) toxins to be active,strongly alkaline conditions are a prerequisite [Bacillus thuringensis,an environmental biopesticide: Theory and Practice, 1993, eds. P. F.Entwistl et al., Wiley & Son, UK]. The guts of the larvae belonging tothe orders of insects known to be sensitive to the Bt toxins compriseenvironmental pools of a high alkaline nature (approx pH 10). Suchinsect orders are especially Isoptera, Lepidoptera, Coleoptera andDiptera.

[0031] Consequently in a preferred embodiment, the pool ofmicroorganisms is isolated from the gut of an insect of the Isoptera,Lepidoptera, Coleoptera, or Diptera families.

[0032] In a more preferred embodiment, the pool of microorganisms isisolated from the gut of insects selected from the group consisting ofAgrotis, Neotermes castaneus, Tineola bisselliella, and Melolonthavulgaris.

[0033] Prior to isolating environmental pools of organisms from animalstomachs or insect guts, it is interesting to do an enrichment byrearing or supplying the animal or insect with food comprising asubstrate for the activity of the polypeptide of interest, maybe even asthe primary carbon and/or nitrogen source. This makes a cow's rumen andlarval guts from Lepidopteran, Coleopteran and Dipteran species highlyinteresting for further enrichment through feeding with specificsubstrates.

[0034] In a preferred embodiment the pool of microorganisms is enrichedby supplying feed to the animal or insect, which comprises a substratefor the polypeptide with an activity of interest.

[0035] Specific examples of “DNA encoding a polypeptide with an activityof interest” include among others enzymatic activity and anti-microbialactivity.

[0036] In a preferred embodiment, the gene libraries are enriched in DNAencoding an enzyme activity of interest.

[0037] In a more preferred embodiment of the invention the activity ofinterest is an enzymatic activity, such as an activity selected from thegroup comprising of phosphatases oxidoreductases (E.C. 1), transferases(E.C. 2); hydrolases (E.C. 3), such as esterases (E.C. 3.1), inparticular lipases and phytase; such as glucosidases (E.C. 3.2), inparticular xylanase, cellulases, hemicellulases, and amylase, such aspeptidases (E.C. 3.4), in particular proteases; lyases (E.C. 4);isomerases (E.C. 5); ligases (E.C. 6).

[0038] In another preferred embodiment, the enzyme of interest comprisesa protease, lipase, beta-galactosidase, lactase, polygalacturonase,beta-glucoamylase, esterase, hemicellulase, peroxidase, oxidase, laccaseor glucose oxidase.

[0039] In more preferred embodiment, the enzymes obtained in the methodis an amylase, in particular an α-amylase or a β-amylase, anarabinanase, an arabinofuranosidase, a galactanase, an α-galactosidase,a β-galactosidase, a polygalacturonase, a pectin methyl esterase, arhamnogalacturonase, a rhamnogalacturon acetyl esterase, a pectin lyase,a xylanase, a cellulase, a β-glucosidase, a cellobiohydrolase, axylosidase, a mannanase, and/or a glucuronisidase.

[0040] The environmental pool of organisms containing DNA encoding apolypeptide with an activity of interest are typically microorganismssuch as Eubacteria, Archaebacteria, fungi, algae and/or protozoa.

[0041] The polypeptide may be an enzyme of interest obtained from anyknown organism. Preferably the enzyme may be obtained frommicroorganism, in particular from a bacteria, from a filamentous fungusor a yeast.

[0042] In the method of the invention, the organisms are enrichedcultures meaning that the cultures are selected on a specific substratefrom which other organisms are not able to grow or having a reducedgrowth.

[0043] It is another object of the invention to provide a method ofselecting a DNA sequence of interest from an environmental pool oforganisms, which method comprises:

[0044] a) subjecting the environmental pool of organisms to cultivationin a medium and/or conditions suitable for enriching said pool oforganisms in organisms harbouring said DNA sequence;

[0045] b) producing gene libraries from the resulting enriched pool oforganisms;

[0046] c) screening the libraries for DNA containing the desired gene;and

[0047] d) selecting the DNA sequence of interest resulting from thescreening of step c).

[0048] Step a) and b) are as described above. In step c), clones foundto comprise a DNA sequence originated from the prepared gene library instep b) may be screened for any activity of interest. Examples of suchactivities include enzymatic activity, anti-microbial activity orbiological activities. In step c), gene libraries are screened forgenomic DNA containing the desired gene, and in step d), the DNAsequence of interest are selected from the screening of step c). Step c)and d) may be performed by standard methods known in the art.

[0049] The polypeptide with the activity of interest may then be testedfor a desired performance under specific conditions and/or incombination with e.g. chemical compounds or agents. The gene librariesmay be screened according to the method of the invention for apolypeptide with an activity of interest e.g. a specific activity,and/or a specific property of interest such as thermostability, high pHtolerance, wash performance, textile dyeing, hair dyeing or bleachingproperties, a effect in feed or food ect. The appropriate assay fortesting for a desired activity and/or property well be known to theskilled person.

[0050] In a preferred embodiment of the method, the gene librarycomprises an enzyme-encoding gene of interest, and the gene library isscreened for enzymes under conditions which the enzyme is active. Thismeans that the library may be screened for enzymes at e.g. hightemperatures such as 60-110° C. and high pH such as 10-12 e.g. in caseswhere it is desired to isolate a DNA sequence encoding an alkalineenzyme with a relatively high thermostability. However, pH can be in anyrange e.g. of from about 0 to about 12, and the temperature in any rangee.g. of from about 5 to about 110° C., preferably of from about 60 toabout 90° C.

[0051] It is still another object of the invention to provide a genelibrary prepared from an environmental pool of organisms enriched in DNAencoding an polypeptide with an activity of interest. In a preferredembodiment, the gene library comprises a polypeptide with an activity ofan enzyme, a hormone or a toxin. In a more preferred embodiment, thegene library comprises an enzyme activity of interest as describedabove.

[0052] The invention is further illustrated in the following exampleswhich is not intended to be in any way limiting to the scope of theinvention.

EXAMPLES Example 1

[0053] Enrichment Procedure

[0054] Shake flasks with 100 ml of the respective media described belowwere inoculated with approximately 1 g of soil samples (NS Collection),and incubated at 60° C. overnight at 250 rpm. The pH in the shake flasksafter incubation were 9.7 to 9.9. All enrichments were checked forgrowth by microscopy.

[0055] Media for enrichment were prepared by mixing of the 10 foldconcentrated stock solutions below: A: KH₂PO₄ 4.25 g/l NH₄Cl 4.25 g/lKCl 4.25 g/l MgSO₄, 7H₂O 6.25 g/l CaCl₂, 2H₂O 3.12 g/l B: NaHCO₃   30g/l Na₂CO₃   30 g/l C: Yeast extract   5 g/l Pectin Pectin 35   20 g/lCellulose CMC C-4888 Sigma   10 g/l Cellulose Powder   20 g/l StarchSoluble starch   50 g/l

[0056] All stock solutions were autoclaved.

[0057] The respective liquid enrichment media were prepared by mixing100 ml of A+B+C+100 ml pectin or cellulose or starch and 600 ml sterilewater.

Example 2

[0058] Enrichment Library Material

[0059] The enrichments were used for preparation of a mixed enrichmentlibrary. 50 ml of each selected enrichment culture were centrifuged andthe combined cell pellets used for gene library construction. The cloneswere obtained by screening of the library, distributed on amylase,arabinase, xylanase, galactanase and pectinase activity.

Example 3

[0060] Preparation of Gene Library No. 1

[0061] The cells from the cultures were washed with 0.9% NaCl and pooledinto one tube.

[0062] DNA was extracted using method described by Pitcher et al.(Pitcher, D. G., saunders, N. A., Owen; R. J. (1989). Rapid extractionof bacterial genomic DNA with guanidium thiocyanate.Lett.Appl.Microbiol., 8, 151-156.). The DNA extraction recovered 170 μghigh molecular weight DNA. Approximately 90 μg DNA in 25% glycerol werefragmented in a nebulizer (Bio Neb Cell Disruption System with an DNAinsert, Glas-Col Apparatus Company) for 45 sec at a pressure of 15 PSI.This resulted in DNA from 2-5 Kb. The DNA was size-fractionated on asucrose gradient (Maniatis et al.), and the fractions of interest werepooled and concentrated by EtOH or isopropanol precipitation. To trimthe ends 4 μg DNA was EtOH precipitated and resuspended in 35 μl H₂O.

[0063] The DNA were trimmed to make blunt ends.

[0064] 35 μl DNA (4 μg)

[0065] 5 μl NEB4 buffer

[0066] 4 μl dNTP (2.5 mM stock)

[0067] 4 μl T4 DNA polymerase

[0068] 2 μl Klenow

[0069] The reaction mixture was incubated at room temp. for 30 min, and200 μl 1×TE, pH 8.0, were added. The mixture was extracted with 1×phenol-chloroform, 1×CIA, 0.1 vols 3 M NaOAc was added, pH 5.2, 2 vols96% EtOH were added, ppt on ice for 30 min, or overnight at −20° C. andresupended in 16 μl H₂O.

[0070] The end trimmed DNA was ligated into a fresh Eco RV digestedpZero (Invitrogen).

[0071] The ligation mixture was transformed into DH10B E. coli cells byelectroporation and frozen in aliqouts corresponding to 300 zeocinresistant colonies. The frozen alliqouts constitute the library no. 1.

Example 4

[0072] Isolation of Bacteria from Soil and Preparation of Gene LibraryNo. 2

[0073] Bacterial isolation from soil was as described by Prieme, A. etal. and Bakken, L. R., (FEMS Microbiology Ecology 21:59-68, 1996). 50 gof soil (obtained from the Roskilde Fjord in Denmark) and 200 ml dH₂Owere blended for 1 min (Waring blender) and placed 1 min on ice. Thiswas repeated 3 times. The suspension was left on the bench for 2 min toallow the large soil particles to settle. 30 ml of the suspension wasadded to a centrifuge tube followed by 10 ml Nycodenz* (*Nycodenz; 0.8g/ml in H₂O, sterile filtered, Nycomed pharma A/S batch no. 207051)which was added via a syringe to the base of the tube. The samples werecentrifuged with a swing-out rotor at 10,000×g for 2 h, no break, 20° C.The bacteria were collected in the interface between the Nycodenz andwater phases (the soil remains were at the base of the tube) and wereremoved with a syringe.

[0074] DNA was extracted using the method described by Pitcher et al.(Pitcher, D. G., saunders, N. A., Owen; R. J. (1989). Rapid extractionof bacterial genomic DNA with guanidium thiocyanate.Lett.Appl.Microbiol., 8, 151-156.).

[0075] The DNA was partially digested with the restriction enzyme Sau3Aand the DNA was size-fractionated on a 1% agarose gel (Maniatis et al.).The agarose containing the DNA corresponding to 3 kb and upwards in sizewas cut from the agarose gel and the DNA was concentrated by furtherelectrophoresis into a 1.2% agarose gel. The DNA was isolated from theagarose piece using the GFX kit (Pharmacia).

[0076] The Sau3A digested DNA was ligated into a fresh BamHI digestedpZero-2 (Invitrogen). The ligation mixture was transformed into DH10B E.coli cells by electroporation and frozen in aliqouts corresponding to300 kanamycin resistant colonies. The frozen aliqouts constitute thelibrary no. 2.

Example 5

[0077] Assaying Gene Library No. 1 for Enzyme Activity

[0078] Amylase Assay

[0079] The assay contains the following reagents:

[0080] 1: 0.1% AZCL Amylose (MegaZyme, Australia)

[0081] 2: 0.1 M Tris-Cl buffer pH9

[0082] 3: MilliQ H2O

[0083] 96 well: In each well were 150 μl (˜15 ml/microplate) used asstandard volume.

[0084] 384 well: In each well were 60 μl (˜25 ml/microplate) used asstandard volume.

[0085] The cells were grown for 65 hours, and thereafter 50 μl of cellswere pipetted into 150 μl assay substrate for 96 well plates or 20 μlinto 60 μl assay substrate for 384 well plates. The assay-plates wereincubated over night at 50° C. in a bag. Positive reactions wereobserved as blue colour in the well.

[0086] Arabinase Assay

[0087] The assay contains the following reagents:

[0088] 1: 0.1% AZCL—Debranched Arabinan (MegaZyme, Australia)

[0089] 2: 0.1 M Tris-Cl buffer pH 9

[0090] 3: MilliQ H20

[0091] 96 well: In each well were 1501 used as standard volume.

[0092] 384 well: In each well were 60 μl used as standard volume.

[0093] The cells were grown for 65 hours, and thereafter 50 μl of cellswere pipetted into 150 μl assay substrate for 96 well plates or 20 μlinto 60 μl assay substrate for 384 well plates. The assay-plates wereincubated over night at 50° C. in a bag. Positive reactions wereobserved as blue colour in the well.

[0094] Galactanase Assay

[0095] The assay contains the following reagents:

[0096] 1: 0.1% AZCL Galactan (MegaZyme, Australia)

[0097] 2: 0.1 M Tris-Cl buffer pH9

[0098] 3: MilliQ H20

[0099] 96 well: In each well were 1501 used as standard volume.

[0100] 384 well: In each well were 601 used as standard volume.

[0101] The cells were grown for 65 hours, and thereafter 50 μl of cellswere pipetted into 150 μl assay substrate for 96 well plates or 20 μlinto 60 μl assay substrate for 384 well plates. The assay-plates wereincubated over night at 50° C. in a bag. Positive reactions wereobserved as blue colour in the well.

[0102] Pectinase Assay

[0103] Numbered black microtiter plates were filled with 150 ml assaymix* as described below using a Multidrop instrument. Subsequently, 50ml of cells were pipetted automatically into the assay plates using aPlate Mate pipetting station. Plates were left at room temperature (inthe dark) for approximately 150-180 min., and subsequently read by aFPM-2 fluorescence polarization reader using excitation-filter 485/22and emission-filter 530/30. Positive clones were scored as a lowering ofthe polarization value which typically was from approximately 90 mP to50-70 mP.

[0104] 150 μl assay mix*

[0105] 34 μg/ml Fluorescein-labelled lemon-pectin 77% DE=5.0 μl 1 g/lsolution 2 mM CaCl2 = 0.5 μl 1 M solution

[0106] 83 vol. % 0.1 M glycin-buffer with 0.1 M NaCl=125 μl buffer pH10.0 Mili-Q-H2O = 20 μl

[0107] Xylanase Assay

[0108] The assay contains the following reagents:

[0109] 1: 0.1% AZCL—Xylan (MegaZyme, Australia)

[0110] 2: 0.1 M Tris-Cl buffer pH9

[0111] 3: MilliQ H20

[0112] 96 well: In each well were 15 μl used as standard volume.

[0113] 384 well: In each well were 60 μl used as standard volume.

[0114] The cells were grown for 65 hours, and thereafter 501 of cellswere pipetted into 150 μl assay substrate for 96 well plates or 20 μlinto 60 μl assay substrate for 384 well plates. The assay-plates wereincubated over night at 50° C. in a bag. Positive reactions wereobserved as blue colour in the well.

[0115] Plate Screening Assay for Xyloglucanase, Galactanase and AmylaseContaining E. Coli Transformants.

[0116] Gene Library no. 2 was screened on LB agar plates containing 25μg/ml kanamycin as the antibiotic selection marker and 0.03%AZCL-xyloglucan+0.03% AZCL-galactan+0.03% AZCL-amylose as enzymesubstrates at 37° C. The formation of a blue halo around the colonyindicates enzyme activity. The colony was restreaked onto LB platescontaining each of the AZCl substrates to identify the enzyme activity.Three amylase positive clones were discovered.

[0117] Positives Obtained from Library No. 1 by Screening with AboveDescribed Assays.

[0118] Three amylase positive clones, two xylanase positive, twopectinase positive, two galactanase positive, and finally eightarabinase positive clones were discovered.

[0119] These results demonstrate that it is possible to select a DNAsequence of interest with the present method of the invention.

Example 6

[0120] Enrichment of Termite Larvae Gut for Cellulases

[0121] Materials: Termite larvae (Neotermes castaneus) were acquiredfrom BAM (Bundesanstalt für Materialforschung und -Prüfung, Berlin,Germany).

[0122] Enrichment procedure: The larvae were subsequently reared and fedon non-sterile plant materials, originating either from gymnosperms orangiosperms (monocot or dicot), enabling enzymatic (endo- and exo-)digestion through plant cell wall degrading enzyme activity.

[0123] Further enrichment through dissection (optional): The larvae weredecapitated under a stereoscopic microscope after which the guts(including gut content) were selected and pooled together from severalanimals.

[0124] DNA preparation: DNA preparations were made from such compositegut materials using commercially available DNA kits (FAST DNA-KitH, Bio101 Inc, 1070 Joshua Way, Calif., US). This high quality DNA materialwas used to prepare a genomic library, e.g. following a protocol asfollows: digestion by Sau3A, fractionation and selection of specificsize range, cloning in the Bacteriphage Lambda-Zap-Express (AHDiagnostic, originating from Stratagene, US). Full protocols are givenby the kit-manufacturers.

[0125] RNA preparation: Total RNA preparations were made from saidcomposite gut materials using commercially available RNA kits and publicprotocols, as e.g. indicated in (H.Dalbøge, 1997, FEMS MicrobiologyReviews 21, 29-42). The mRNA fraction was subsequently harvested. Basedon this fraction, the corresponding cDNA prep was made. This was used toconstruct a cDNA library, representing the expressed proteins at thegiven time. Protocols and references for mRNA, cDNA and cDNA libraryconstruction are available in common textbooks (plus in e.g. H.Dalbøge,1997, FEMS Microbiology Reviews 21: 29-42).

[0126] Screening of the genomic library enriched for DNA from thoseorganisms that specifically benefitted from the feeding conditions ofthe larvae under preparation: A plaque screening procedure was adaptedto function with enzyme substrate holding plates (e.g. prepared from theAZCL blue granule substrates, available from MegaZyme). Thuscolour-halos indicated which phages were holding an inserted fullfunctional gene, encoding an enzyme with the cellulase activity ofinterest. The procedure is most successful when the enzyme substrate isincorporated in a bottom layer and the phages are added in a separatelayer on top. The positive plaques on the AZCL substrate plates could bedetected by their blue halos.

[0127] Screening of the cDNA library enriched for high expression ofproteins useful to degrade the feed given to the larvae was performedaccording to the protocol for expression cloning, as given in H.Dalbøge,1997 (FEMS Microbiology Reviews 21: 29-42).

[0128] Identified hits: more than 200 cellulase active clones werefinally identified on HE Azur cross linked blue granule substrates fromMegaZyme. PCR made directly from individual colonies (PCR procedures asindicated in relevant text books, using e.g. the polymerases availablefrom Advanced Biotechnologies, Surrey, UK) was used to differentiate andgroup the hits. The primers used were based on recognition andamplification of the sense and antisense cDNA cloning plasmid pYes-2(commercially available from Invitrogene, US).

[0129] At least four different sized functional genes were herebyidentified.

Example 7

[0130] Enrichment of Textile Moth Larvae Gut for Proteases

[0131] Materials: Larvae of Tineola bisselliella, the lepidopterantextile moth were acquired from BAM in Germany. Standard protocols arereferred in Example 6.

[0132] Enrichment procedure: The larvae were subsequently reared and fedon non-sterile protein-rich materials (as e.g. feather, hair and wool).

[0133] Further enrichment through dissection (optional): The larvae weredecapitated under a stereoscopic microscope after which the gut(including gut content) was selected and pooled together from severalanimals

[0134] DNA preparation: DNA preps were made from such composite gutmaterials using commercially available DNA kits. This high quality DNAmaterial was used to prepare a genomic library.

[0135] RNA preparation: Total RNA preps were made from said compositegut material using commercially available RNA kits and protocols. ThemRNA fraction was subsequently harvested. Based on this thecorresponding cDNA prep was made. This was used to construct a cDNAlibrary, representing the expressed proteins at the given time.

[0136] Screening of the genomic library enriched for DNA from thoseorganisms specifically benefiting from the feeding conditions of thelarvae under preparation: a plaque screening procedure was adapted toenzyme substrate holding plates, indicating which phages were holding aninsert of a full functional gene encoding an enzyme with the proteaseactivity of interest.

[0137] Screening of the cDNA library enriched for high expression ofproteins useful to degrade the feed given to the larvae: the protocolfor expression cloning, as given in H.Dalbøge, 1997 (FEMS MicrobiologyReviews 21, 29-42).

[0138] Identified hits: Protease active clones could be identified byscreening on substrate plates with AZCL-casein blue granules fromMegaZyme. The protease hits could be further subdivided according towhich types of protein bonds they specifically degrade.

Example 8

[0139] Enrichment of Melolontha Vulgaris Larvae Gut for Plant Cell WallDegrading Enzymes

[0140] Materials: Larvae of Melolontha vulgaris (Coleoptera) werecollected from Danish habitats (Zealand) where the soil is rich in avery varied composition of plant debris. The larvae of this species arefree living in soil, feeding for up to 3 years on plant materials

[0141] Enrichment procedure: The larvae were subsequently reared and fednon-sterile non-specified plant debris of a very broad taxonomiccomposition.

[0142] Further enrichment through dissection (optional): The larvae weredecapitated under a stereoscopic microscope after which the guts(including gut content) were selected and pooled together from severalanimals.

[0143] DNA preparation: DNA preps were made from such composite gutmaterial using commercially available DNA kits. This high quality DNAmaterial was used to prepare a genomic library.

[0144] RNA preparation: Total RNA preps were made from said compositegut material using commercially available RNA kits and protocols. ThemRNA fraction was subsequently harvested. Based on this, thecorresponding cDNA prep was made. This was used to construct a cDNAlibrary, representing the expressed proteins at the given time.

[0145] Screening of the genomic library enriched for DNA from thoseorganisms specifically benefiting from the feeding conditions of thelarvae under preparation: a plaque screening procedure was adapted toenzyme substrate holding plates, indicating which phages were holding a(functional) gene insert encoding an enzyme with the plant cell walldegrading activity of interest.

[0146] Screening of the cDNA library enriched for high expression ofproteins useful to degrade the feed given to the larvae: the protocolfor expression cloning, as given in H.Dalbøge, 1997 (FEMS MicrobiologyReviews 21, 29-42).

[0147] Identified hits: Numerous types of cell wall degrading enzymescould be identified by screening on the variety of Azur cross linkedblue granule substrates available from MegaZyme.

Example 9

[0148] Enrichment of Agrotis Larvae Guts for Amylases

[0149] Materials: Larvae of Agrotis (Lepidoptera) were received fromRVAU (Royal Veterinary and Agricultural University), Copenhagen(Professor Peter Esbjerg).

[0150] Enrichment procedure: The larvae were subsequently reared and fednon-sterile starch rich materials.

[0151] Further enrichment through dissection (optional) the larvae weredecapitated under a stereoscopic microscope after which the guts(including gut content) were selected and pooled together from severalanimals.

[0152] DNA preparation: DNA preps were made from such composite gutmaterial using commercially available DNA kits. This high quality DNAmaterial was used to prepare a genomic library.

[0153] RNA preparation: Total RNA preps were made from the composite gutmaterial using commercially available RNA kits and protocols. The mRNAfraction was subsequently harvested. Based on this fraction thecorresponding cDNA prep was made. This was used to construct a cDNAlibrary, representing the expressed proteins at the given time.

[0154] Screening of the genomic library enriched for DNA from thoseorganisms specifically benefitting from the feeding conditions of thelarvae under preparation: a plaque screening procedure was adapted toenzyme substrate holding plates, indicating which phages were holding aninsert of a functional gene, encoding an enzyme with a plant cell walldegrading activity of interest.

[0155] Screening of the cDNA library enriched for high expression ofproteins useful to degrade the feed given to the larvae: the protocolfor expression cloning, as given in H.Dalbøge, 1997 (FEMS MicrobiologyReviews 21, 29-42).

[0156] Identified hits: The AZCL-amylose blue granule substrates wereused for a plate screening of the yeast (and the plaque) colonies. Highalkaline amylases could be found by overlaying the expression cloningyeast plates with glycine buffer, pH 10. The colonies which onlydeveloped the blue halo of diffusing blue colour around the granulesafter buffer treatment, were the clones in which a gene encoding analkaline amylase had been inserted and was expressed.

Example 10

[0157] Enrichment of Cow Rumen Content for Cellulases, Proteases andAmylases

[0158] Materials: Samples were taken in a semianaerobic manner directlyfrom a fistulated cow (at RVAU, Rørrendeg{dot over (a)}rd, T{dot over(a)}strup, Denmark).

[0159] Enrichment procedure: The cow was in the weeks prior to thesampling fed material of specific composition e.g. hey to enrich forcellulase and other plant cell wall degrading enzymes, cereal grains toenrich for amylase activity and soy to enrich for protease activities.

[0160] Further enrichment through dissection (optional): Microscopicanalysis was made on further dissected fractions, reflecting the variousdegrees of feed breakdown in the rumen.

[0161] DNA preparation: DNA preps were made from such composite rumenmaterial using commercially available DNA kits. This high quality DNAmaterial was used to prepare a genomic library.

[0162] RNA preparation: Total RNA preps were made from the compositerumen material using commercially available RNA kits and protocols. ThemRNA fraction was subsequently harvested. Based on this thecorresponding cDNA prep was made. This was used to construct a cDNAlibrary, representing the expressed proteins at the given time.

[0163] Screening of the genomic library enriched for DNA from thoseorganisms specifically benefitting from the feeding conditions of thelarvae under preparation: a plaque screening procedure was adapted to bemade on enzyme substrate holding plates, indicating which phages wereholding an insert of a full functional gene, encoding an enzyme with theactivity of interest.

[0164] Screening of the cDNA library enriched for high expression ofproteins useful to degrade the feed given to the larvae: the protocolfor expression cloning, as given in H.Dalbøge, 1997 (FEMS MicrobiologyReviews 21, 29-42).

[0165] Identified hits: Several types of enzyme activities were found,e.g. more than 20 cellulase active clones were identified on HE Azurcross linked blue granule substrates from MegaZyme. Colony PCR was usedto differentiate and group the hits. At least four different sizedfunctional genes were hereby identified.

1. A method for generating a gene library from an environmental pool ofmicroorganisms, which gene library is enriched in DNA encoding apolypeptide with an activity of interest, which method comprises: a)subjecting the environmental pool of microorganisms to cultivation underconditions wherein the pool of microorganisms is enriched inmicroorganisms harbouring said DNA, thereby forming an enrichedenvironmental pool of microorganisms, and b) preparing a gene libraryfrom the enriched environmental pool of microorganisms, wherein prior tosaid preparing there is no further purification of the enrichedenvironmental poor of microorganisms.
 2. The method of claim 1, whereinthe conditions are culturing in a medium that contains a substrate forthe polypeptide with an activity of interest encoded by said DNA.
 3. Themethod of claim 2, wherein the substrate constitutes the carbon sourceand/or nitrogen source of the medium.
 4. The method of claim 2, whereinthe substrate comprises pectin, amylose, cellulose, galactose, xylose orarabinose or a combination thereof.
 5. The method of claim 1, whereinthe pool of microorganisms is enriched by one or more growthrestrictions.
 6. The method of claim 5, wherein the growth restrictionscomprise pH and temperature.
 7. The method of claim 5, wherein thegrowth restrictions are pH 9-11 and temperature 50-70° C.
 8. The methodof claim 1, wherein the gene library is enriched in DNA encoding anenzyme of interest.
 9. The method of claim 8, wherein the enzyme ofinterest comprises a hydrolase, an oxidoreductase, a transferase, alyase or a ligase.
 10. The method of claim 8, wherein the enzyme ofinterest comprises a protease, lipase, beta-galactosidase, lactase,polygalacturonase, beta-glucoamylase, esterase, hemicellulase,peroxidase, oxidase, laccase or glucose oxidase.
 11. The method of claim8, wherein the enzyme of interest is a pectinase, an amylase, agalactanase, an arabinase, a xylanase, or a cellulase.
 12. The method ofclaim 1, wherein the environmental pool of microorganisms comprisesenzyme producing microorganisms.
 13. The method of claim 1, wherein themicroorganisms comprise bacteria or fungi.
 14. A method of identifying aDNA sequence encoding a polypeptide of interest from an environmentalpool of microorganisms, which method comprises: a) subjecting theenvironmental pool of microorganisms to cultivation under conditionswherein the pool of microorganisms is enriched in microorganismsharbouring said DNA sequence, thereby forming an enriched environmentalpool of microorganisms; b) producing gene libraries from the enrichedenvironmental pool of microorganisms, wherein prior to said preparingthere is no further purification of the enriched environmental poor ofmicroorganisms, and c) screening the libraries of step b) to identify aDNA sequence encoding the polypeptide of interest.
 15. The method ofclaim 14, wherein the polypeptide of interest encodes an enzyme.
 16. Themethod of claim 14, wherein the gene libraries are screened in step c)for an active enzyme.
 17. The method of claim 14, wherein thepolypeptide of interest encodes a hydrolase, an oxidoreductase, atransferase, a lyase or a ligase.
 18. The method of claim 14, whereinthe polypeptide of interest encodes a protease, lipase,beta-galactosidase, lactase, polygalacturonase, beta-glucoamylase,esterase, hemicellulase, peroxidase, oxidase, laccase or glucoseoxidase.
 19. The method of claim 14, wherein the polypeptide of interestencodes a pectinase, an amylase, a galactanase, an arabinase, axylanase, or a cellulase.
 20. The method of claim 14, wherein theconditions are culturing in a medium that contains a substrate for thepolypeptide with an activity of interest encoded by said DNA.